Macrophage mitochondrial bioenergetics and tissue invasion are boosted by an Atossa‐Porthos axis in Drosophila

Abstract Cellular metabolism must adapt to changing demands to enable homeostasis. During immune responses or cancer metastasis, cells leading migration into challenging environments require an energy boost, but what controls this capacity is unclear. Here, we study a previously uncharacterized nuclear protein, Atossa (encoded by CG9005), which supports macrophage invasion into the germband of Drosophila by controlling cellular metabolism. First, nuclear Atossa increases mRNA levels of Porthos, a DEAD‐box protein, and of two metabolic enzymes, lysine‐α‐ketoglutarate reductase (LKR/SDH) and NADPH glyoxylate reductase (GR/HPR), thus enhancing mitochondrial bioenergetics. Then Porthos supports ribosome assembly and thereby raises the translational efficiency of a subset of mRNAs, including those affecting mitochondrial functions, the electron transport chain, and metabolism. Mitochondrial respiration measurements, metabolomics, and live imaging indicate that Atossa and Porthos power up OxPhos and energy production to promote the forging of a path into tissues by leading macrophages. Since many crucial physiological responses require increases in mitochondrial energy output, this previously undescribed genetic program may modulate a wide range of cellular behaviors.


4th Aug 2021 1st Editorial Decision
Dear Dr. Siekhaus, Thank you for submitting your manuscript entitled "A genetic program boosts mitochondrial function to power macrophage tissue invasion" [EMBOJ-2021-109049] to The EMBO Journal. Your study has now been assessed by three reviewers, whose reports are enclosed below.
As you can see, the referees concur with us on the potential interest of your findings. However, they also raise major critical points that need to be addressed before they can support publication here. In particular, referee #2 states that one of the key conclusions of the paper -i.e. Porthos regulates mRNA translation -is only based on polysome-Seq analysis. Therefore, this referee requests you to perform RT-qPCR analyses of selected (and representative) mRNAs identified as being translationally repressed across the polysome gradient. In addition, s/he asks you to show that protein levels encoded by such mRNAs are also altered. Referee #3 finds that additional mechanistic data are needed to support the main claims and asks you to investigate i) how LKR/SDH and GR/HPR regulate migration and mitochondrial bioenergetics; and ii) how Porthos/40S ribosome assembly defects affect nuclear-encoded mitochondrial gene translation. Furthermore, both referee #2 and #3 request you to test whether TORC1 activity is affected in Athos and Porthos deficient cells.
Given the overall interest of your study, I am pleased to invite submission of a manuscript revised as indicated in the reports attached herein. I would like to point it out that addressing all referees' points in a conclusive manner, as well as a strong and unanimous support from the reviewers, would be essential for publication in The EMBO Journal. I should also add that it is our policy to allow only a single round of major revision. Therefore, acceptance of your manuscript will depend on the completeness of your responses in this revised version.
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Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide The revision must be submitted online within 90 days; please click on the link below to submit the revision online before 2nd Nov 2021. https://emboj.msubmit.net/cgi-bin/main.plex This is a beautiful study from Emtanani and colleagues that marries live imaging with the powerful genetics of Drosophila to dissect a novel mechanism by which immune cells are able to increase mitochondrial function to fuel invasive migration in vivo. By studying a subset of macrophages that carry out a characteristic invasive migration into the germ band of the developing embryo the authors identify the previously unstudied nuclear protein -Atossa as a key regulator of this invasive migration. They go on to tease apart the mechanism by which Atossa and a downstream target Porthos together increase the efficiency and amount of OxPhos within the macrophages to produce sufficient ATP to power tissue invasion. The manuscript is well written, the data is of extremely high quality throughout and support all the claims made in the paper. The findings are novel and high impact and the paper is highly appropriate for publication in The EMBO journal. The only question I would suggest the authors need to address is why atossa expression is maintained at a high level in all macrophages during stages 9-12 when it is only required within the first two pioneer macrophages in order to drive invasive migration into the germ band? Is this shift in metabolism required for or enhance other macrophage behaviours during development such as apoptotic cell clearance? Whilst this might be beyond the scope of this study to show experimentally, the authors should include in the discussion at least.

Referee #2:
Here the authors present a manuscript investing metabolic dynamics in the Drosophila immune system that govern how macrophages migrate and invade into the extended germband during development. They identify CG9005, which they rename Atossa, as being critical for early steps in pioneer macrophage invasion of the germband. They go on to characterize Atossa, find that it is a nuclear protein that appears to be a transcription factor. Importantly, they demonstrate that Atossa mutants lacking trans-activating domains does not rescue defects in Atossa deficient embryos, but that embryos can be rescued by expressing the murine ortholog mFAM214-A-B. RNA-Seq analysis of Atossa deficient macrophages identified a number of differentially expressed genes, with many downregulated genes being linked to mitochondrial dynamics (redox), as well as a gene coding for a predicted ATP-dependent RNA helicase (CG9253) that they name Porthos. They go on to show that Porthos localizes to both the nucleus and the nucleolus and show that knocking down Porthos also leads to a defect in macrophage migration into the germband. Based on its nucleolar localization, they carried out polysome profiling to investigate if Porthos impacts mRNA translation. A decrease in heavy polysomes and a concomitant increase in 60S subunit was observed in Porthos depleted cells. Polysome-Seq identified a number of genes that were translationally repressed, including many involved in mitochondrial respiration. Along these lines, they find that Porthos is required for mitochondrial respiration and energy production in order initiate germband invasion.
Overall, while I am not an expert in Drosophila development, I find that the paper is well-written, and the data clear and convincing for the most part. A major conclusion of the paper is that Porthos regulates mRNA translation. However, this is currently only based on polysome-Seq analysis. This should be subsequently validated by carrying out RT-qPCR of select mRNAs that they identify as being translationally repressed across the polysome gradient. This would allow the authors to see if they mRNA distribution does in fact shift from heavier fractions to lower ones. Finally, it would also be important to show that if translation is in fact being altered, that the levels of proteins encoded by these select mRNAs is also being altered. Mitochondrial dynamics are also tightly regulated by TORC1 signaling. Are they in a position to see whether TORC1 status (4E-BP protein phosphorylation) is being impacted in Porthos deficient cells? This could also potentially lead to a similar impact in mRNA translation of nuclear-encoded mitochondrial mRNAs. Finally, while a minor issue...on line 270: 'enhanced translational efficiency of a subset of proteins'. It should be of a subset of mRNAs, not proteins.

Introduction
In this manuscript, Emtenani et al characterize novel regulators of cellular migration using Drosophila macrophages as a model system. The authors characterize the gene CG9005/Atossa, which is enriched in macrophages before germband entry. RNA sequencing analyses uncovered a role of Atossa in driving gene expression of redox and stress responsive genes. Of the 5 most downregulated genes in Atossa mutants, 3 were required for germband migration including Porthos, a DEAD-box RNA helicase localized to the nucleolus and two metabolic enzymes LKR/SDH and GR/HPR. Density gradient fractionation experiments in S2R+ cells confirmed that Porthos deficiency leads to defects in ribosomal assembly and cytosolic translation. Ribosomal profiling experiments found 204 coding genes were less efficiently translated, including many mitochondrial genes, and 102 were more efficiently translated in Porthos mutants. The authors conclude that reduced translation of nuclear-encoded mitochondrial mRNAs from Atossa or Porthos deficiency causes mitochondrial dysfunction and prevents effective path invasion by leader cells.
This manuscript is interesting and so is their observation that ATP/mitochondrial metabolism is important for initial tissue invasion events by macrophages rather than general migration speed or directionality. Their overall conclusions need additional data to support the main claims, there are some mechanistic details missing from their manuscript, and at this point it seems preliminary. Namely, i) how LKR/SDH and GR/HPR regulate the migration phenotype is largely speculative and assumed to be related to mitochondrial bioenergetics and ii) how Porthos/40S ribosome assembly defects affect nuclear-encoded mitochondrial gene translation.
Major comments: • Please determine if there are mitochondrial respiratory defects in LKR/SDH and GR/HPR mutants to solidify the connection of these genes to respiratory capacity. It is possible that these proteins do not play a role in mitochondrial respiratory capacity and ATP generation, and their role on migration is independent. • There needs to be a better rationale of why ribosomal biogenesis defects cause specific mitochondrial defect instead of widespread issues with protein synthesis? Are the identified targets enriched for TISU elements that require cap-dependent translation? mRNAs with these structures usually encode mitochondrial proteins and are sensitive to mTORC1 inhibition. Is there strong overlap with your data set and others that study these mRNAs? See: 10.1016/j.cmet.2015.02.010 or 10.1101/gr.197566.115 This is an important point, and it is not clear how specific the translation is to mitochondrial proteins, as for example it has been described with the CLUH protein.
• Please determine if mTORC1 signaling (p-4EBP1) is suppressed during Atossa and Porthos LOF. This could help explain the specific defect in nuclear-encoded mitochondrial gene translation based mentioned above. • In the introduction, there is no general principles that elevated mitochondria or PGC1s correlate with invasion, in fact in certain tumors high PGC1a and high mitochondria and oxidative phosphorylation correlate negatively with metastasis, as described by Carracedo's group in Nature Cell Biology (2016) and Cancer Research (2019).
Minor comments: • In the context of Pths rescue in Atossa mutants, can you rescue any molecular phenotypes, e.g. translation defects, mitochondrial respiration, etc.
• Also, please verify that Pths overexpression is working in the Atossa mutants through qPCR or Western.
• As an alternative approach, can you rescue the ribosomal assembly defect in Atossa mutants to determine if this is responsible for downstream mitochondrial phenotypes? One could activate mTORC1 (pharmacological or genetic) to rescue mitochondrial defects present in Atossa mutants since mTORC1 controls ribosome assembly, mitochondrial gene translation, and PGC-1a activity. Or perhaps overexpress a ribosomal assembly factor that promotes 40S assembly. • Can you express a mutant of Pths in Pths KD cells that fails to localize to the nucleus to assess whether its function in nucleolar ribosomal assembly is important for downstream phenotypes? • Fig 6. b,c -For graphical representation of OCR in bar graph form, I would subtract non-mitochondrial respiration as a baseline correction. On that note, it is odd that your non-mitochondrial respiration is so different between genotypes. Perhaps this indicates a seeding/measurement issue since the cells are semi-adherent. • Perform either SDS or BN-PAGE analyses for Atessa and Porthos LOFs to confirm nuclear-encoded translation defects. • Please confirm efficacy of complex III and IV knockdowns by qPCR or Western.
• Is there any mRNA-mRNA or protein-protein correlation between Atossa and PGC-1a? Is there any overlap between Atossa and PGC1-a targets? This could strengthen arguments that these pathways can act in concert.

Reviewer 1)
This is a beautiful study from Emtenani and colleagues that marries live imaging with the powerful genetics of Drosophila to dissect a novel mechanism by which immune cells are able to increase mitochondrial function to fuel invasive migration in vivo. By studying a subset of macrophages that carry out a characteristic invasive migration into the germ band of the developing embryo the authors identify the previously unstudied nuclear protein -Atossa as a key regulator of this invasive migration. They go on to tease apart the mechanism by which Atossa and a downstream target Porthos together increase the efficiency and amount of OxPhos within the macrophages to produce sufficient ATP to power tissue invasion. The manuscript is well written, the data is of extremely high quality throughout and support all the claims made in the paper. The findings are novel and high impact and the paper is highly appropriate for publication in The EMBO journal.
We greatly thank the reviewer for their compliments about our work and its impact.
1. The only question I would suggest the authors need to address is why atossa expression is maintained at a high level in all macrophages during stages 9-12 when it is only required within the first two pioneer macrophages in order to drive invasive migration into the germband? 2. Is this shift in metabolism required for or enhance other macrophage behaviours during development such as apoptotic cell clearance? Whilst this might be beyond the scope of this study to show experimentally, the authors should include in the discussion at least. This is a very interesting question, and studying the effects of Atossa on immune responses is something we seek to tackle in the future. In this work, as the reviewer suggested, we added a section to the discussion in lines 564-570: "At least at the RNA level Atossa is upregulated not just in the first two pioneer macrophages but in all of them. This may enable a large potential pool of macrophages to be capable of serving as the pioneers. Atossa may also support other energy intensive tasks such as apoptotic cell phagocytosis, (Borregaard and Herlin, 1982), a capacity carried out by most migrating macrophages to aid development (Tepass et al., 1994) which also primes their inflammatory responses (Weavers et al., 2016)." …………………………………………………………………………….

Reviewer 2)
Here the authors present a manuscript investing metabolic dynamics in the Drosophila immune system that govern how macrophages migrate and invade into the extended germband during development. They identify CG9005, which they rename Atossa, as being critical for early steps in pioneer macrophage invasion of the germband. They go on to characterize Atossa, find that it is a nuclear protein that appears to be a transcription factor. Importantly, they demonstrate that Atossa mutants lacking trans-activating domains does not rescue defects in Atossa deficient embryos, but that embryos can be rescued by expressing the murine ortholog mFAM214-A-B. RNA-Seq analysis of Atossa deficient macrophages identified a number of differentially expressed genes, with many 21st Jan 2022 1st Authors' Response to Reviewers downregulated genes being linked to mitochondrial dynamics (redox), as well as a gene coding for a predicted ATP-dependent RNA helicase (CG9253) that they name Porthos. They go on to show that Porthos localizes to both the nucleus and the nucleolus and show that knocking down Porthos also leads to a defect in macrophage migration into the germband. Based on its nucleolar localization, they carried out polysome profiling to investigate if Porthos impacts mRNA translation. A decrease in heavy polysomes and a concomitant increase in 60S subunit was observed in Porthos depleted cells. Polysome-Seq identified a number of genes that were translationally repressed, including many involved in mitochondrial respiration. Along these lines, they find that Porthos is required for mitochondrial respiration and energy production in order initiate germband invasion.
Overall, while I am not an expert in Drosophila development, I find that the paper is wellwritten, and the data clear and convincing for the most part.
We very much appreciate the reviewer's positive feedback on the manuscript. We are also grateful to the reviewer for their thoughtful and critical reading of our manuscript, for the suggestions to improve it and for their questions. Addressing these points has greatly strengthened our study.

1.
A major conclusion of the paper is that Porthos regulates mRNA translation. However, this is currently only based on polysome-Seq analysis. This should be subsequently validated by carrying out RT-qPCR of select mRNAs that they identify as being translationally repressed across the polysome gradient.
To validate our polysome profiling data we conducted RT-qPCR analysis on polysome profile fractions for a number of Porthos target transcripts in the control and porthos-KD S2R+ cells. We have assessed the fractions including RNP, 40S/60S, monosome, low polysome (di-and trisome), high polysome (remaining fractions) for 3 independent biological replicates. We normalized the data to the monosome fraction for each transcript.
We observed statistically significant decreases in the mRNA levels on high polysomes for mitochondrial OxPhos complexes I, III and V ( Fig. 6C-E) and strong decreases in the light polysome fractions in porthos-KD S2R+ cells compared to the control. As our collaborator was moving his laboratory we were unable to obtain more samples to increase the significance further. We did not find a similar effect in these fractions when we tested GAPDH gene as a non-target control (Fig. 6F). Thus this RT-qPCR analysis confirms our previous polysome profiling results.
We present these results in lines 348-363 and show the data in Fig 6C-F.

2.
Finally, it would also be important to show that if translation is in fact being altered, that the levels of proteins encoded by these select mRNAs is also being altered.
Unlike in mammalian systems, there are few available antibodies against Drosophila proteins. Thus we were unable to obtain antibodies corresponding to any of the direct targets we identified in the RNAseq. To get around this we utilized commercially available antibodies that had been validated for Drosophila (Teixeira et al 2015 PMID 25915123) against subunits of the OxPhos complexes whose protein levels had been shown to depend on the protein levels of our direct targets. Lower levels of the mammalian ortholog of the predicted complex I assembly factor we identified as a target in the RNAseq lead to reduced levels of other complex I proteins, including MT-ND1 (see Formosa et al., 2015 Fig 2C,D). Similarly, in humans the absence of subunit g of complex V, one of our targets, has been shown to lead to lower protein levels of multiple other subunits including ATP synt-β (see He et al., 2018 Fig 5A). In Western blots we found 73% and 31% lower levels of these CI and CV proteins respectively in pths-KD S2R+ cells compared to the control (CI MT-ND1, p<0.0001; CV ATP synt-β, p=0.03) (shown in Fig 6G-H). To test for a possible general deficiency in protein translation we examined the non-target proteins profilin and tubulin b and found no significant change in levels ( Fig 6I) (profilin, p=0.26; βtubulin, p=0.55). In sum, our results argue that Pths does not affect protein translation generally, but is required for the enhanced levels of a subset of proteins, many of which are involved in mitochondrial and metabolic function.
The results are presented in lines 364-382 and shown in Fig 6G-I. 3. Mitochondrial dynamics are also tightly regulated by TORC1 signaling. Are they in a position to see whether TORC1 status (4E-BP protein phosphorylation) is being impacted in Porthos deficient cells? This could also potentially lead to a similar impact in mRNA translation of nuclear-encoded mitochondrial mRNAs.
To address this and a related question from Reviewer #3 we assessed the activity of the dTORC1 signaling pathway examining the phosphorylation status of the TORC1 kinase target 4EBP1 in the control and porthos-KD S2R+ cells via Western Blot. We saw no significant change in the levels of p-4EBP1 in porthos-KD cells compared to the control (Fig. 5I), arguing that Atossa and Porthos affect the mRNA translation of a set of nuclearencoded mitochondrial mRNAs through a mechanism independent of general translation controlled by dTORC1. We present this data in lines 329-335 and Fig 5I. 4. Finally, while a minor issue...on line 270: 'enhanced translational efficiency of a subset of proteins'. It should be of a subset of mRNAs, not proteins.
We greatly thank the reviewer for catching our error. We have corrected the text accordingly in the abstract and changed the wording in previous line 270 to "is required for the enhanced levels of a subset of proteins" in current line 381 as we have now added more data to substantiate this conclusion.

Introduction
In this manuscript, Emtenani et al characterize novel regulators of cellular migration using Drosophila macrophages as a model system. The authors characterize the gene CG9005/Atossa, which is enriched in macrophages before germband entry. RNA sequencing analyses uncovered a role of Atossa in driving gene expression of redox and stress responsive genes. Of the 5 most downregulated genes in Atossa mutants, 3 were required for germband migration including Porthos, a DEAD-box RNA helicase localized to the nucleolus and two metabolic enzymes LKR/SDH and GR/HPR. Density gradient fractionation experiments in S2R+ cells confirmed that Porthos deficiency leads to defects in ribosomal assembly and cytosolic translation. Ribosomal profiling experiments found 204 coding genes were less efficiently translated, including many mitochondrial genes, and 102 were more efficiently translated in Porthos mutants. The authors conclude that reduced translation of nuclear-encoded mitochondrial mRNAs from Atossa or Porthos deficiency causes mitochondrial dysfunction and prevents effective path invasion by leader cells.
This manuscript is interesting and so is their observation that ATP/mitochondrial metabolism is important for initial tissue invasion events by macrophages rather than general migration speed or directionality. Their overall conclusions need additional data to support the main claims, there are some mechanistic details missing from their manuscript, and at this point it seems preliminary.
Namely, i) how LKR/SDH and GR/HPR regulate the migration phenotype is largely speculative and assumed to be related to mitochondrial bioenergetics? ii) how Porthos/40S ribosome assembly defects affect nuclear-encoded mitochondrial gene translation?
We thank the reviewer for their positive comments on our study. We are also very grateful for the extremely thoughtful questions about the manuscript and the suggestions which we have implemented that have greatly improved the work.

Major comments:
• Please determine if there are mitochondrial respiratory defects in LKR/SDH and GR/HPR mutants to solidify the connection of these genes to respiratory capacity. It is possible that these proteins do not play a role in mitochondrial respiratory capacity and ATP generation, and their role on migration is independent.
We were unable to obtain knockout cell lines for Seahorse analysis. Thus to assess if Atossa's target metabolic enzymes GR/HPR and LKR/SDH can boost the cellular bioenergetics required for macrophage tissue invasion, we tested if overexpressing either of them in macrophages could rescue the bioenergetic phenotype seen in atos PBG macrophages, measuring the pPDH/PDH ratios as a reporter of utilization of the TCA cycle and indirect measure of TCA cycle produced ATP/ADP levels (Fig 8A-B). We simultaneously examined if Porthos, which we have shown affects mitochondrial bioenergetics could rescue the atos PBG phenotype (also to address a later query of this reviewer). Interestingly we observed significantly higher pPDH/PDH ratios in atos PBG macrophages expressing any of the three Atos targets, Porthos, GR/HPR or LKR/SDH, compared to atos PBG mutant embryos (Fig 8A-B). This result strongly supports the conclusion that Atos acts through each of its downstream metabolic proteins to elevate cellular bioenergetics in macrophages. We hypothesize that overexpressing either of these metabolic enzymes which lie upstream of the synthesis of glucose/ Acetyl CoA respectively leads to more fuel being sent into the TCA cycle, compensating for the reduced translation of OxPhos complex components found in the atossa mutant due to the lower levels of Porthos. If this is the case we would predict that expressing GR/HPR and LKR/SDH in macrophages could also rescue their germband invasion defect in atos PBG embryos. They each could rescue (Fig 8C-D), strongly suggesting that GR/HPR and LKR/SDH enzymes act by increasing cellular energetics and ATP generation to facilitate tissue invasion. We also tested if Atossa's murine orthologs mFAM214A and B could rescue the pPDH/PDH levels to see if the capacity to regulate this bioenergetic pathway was conserved in vertebrates and found that they could (Fig 8E-F).
These results are discussed in the manuscript in lines 476-494 and the data presented in Fig 8. • There needs to be a better rationale of why ribosomal biogenesis defects cause specific mitochondrial defect instead of wide-spread issues with protein synthesis? Are the identified targets enriched for TISU elements that require cap-dependent translation? mRNAs with these structures usually encode mitochondrial proteins and are sensitive to mTORC1 inhibition. Is there strong overlap with your data set and others that study these mRNAs? See: 10.1016/j.cmet.2015.02.010 or 10.1101/gr.197566.115 This is an important point, and it is not clear how specific the translation is to mitochondrial proteins, as for example it has been described with the CLUH protein.
To our knowledge TISU elements have not been identified in Drosophila. Through an unbiased search we did not find any significantly enriched motifs from the 5'UTRs of the identified target genes. To determine if the target genes we identified for Porthos contain the TISU sequence we compared their orthologs to the list of TISU containing genes in Sinvani et. al. We hypothesize that Porthos' ability to enhance the translation of the Porthos targets we detected may be due to their shorter 5'UTRs.
These findings are described in the paper in the results in lines 303-312, the bioinformatics analysis approach in the methods in lines 1056-1071 and the 5'UTR length data in Fig 5D.

Reviewer Figure: A
Porthos polysome targets whose orthologs in humans contain TISU sequences. B-C Shared targets between Porthos and CLUH targets shown in the two papers. D

Analysis of 5' UTR length in mRNAs expressed in S2R+ cells and in Porthos targets
• Please determine if mTORC1 signaling (p-4EBP1) is suppressed during Atossa and Porthos LOF. This could help explain the specific defect in nuclear-encoded mitochondrial gene translation based mentioned above.
To address this and a related request from Reviewer #2 we assessed the activity of dTORC1 signaling pathway, examining the phosphorylation status of the TORC1 kinase target 4EBP1 via Western Blot (Fig. 5I). We saw no significant alteration in the levels of p-4EBP1 in porthos-KD S2R+ cells compared to the control (Fig. 5I), arguing that Atossa and Porthos do not upregulate dTORC1 signaling to affect the mRNA translation of a set of nuclear-encoded mitochondrial mRNAs. We describe this data in lines 329-335 and show it in Fig 5I. • In the introduction, there is no general principles that elevated mitochondria or PGC1s correlate with invasion, in fact in certain tumors high PGC1a and high mitochondria and oxidative phosphorylation correlate negatively with metastasis, as described by

Minor comments:
• In the context of Pths rescue in Atossa mutants, can you rescue any molecular phenotypes, e.g. translation defects, mitochondrial respiration, etc.
We observed higher pPDH/PDH ratios in atos PBG macrophages expressing Porthos ( Fig  8B,D) (described in lines 476-compared to atos PBG , arguing that Porthos can restore mitochondrial respiration, and thus enhance cellular energy levels, fitting with its ability to rescue macrophage migration into the germband which we had showed in the original version of the paper in Fig 4I-J. As we do the Pths rescue of the atos PBG phenotype in macrophages, assessing translational effects would require Westerns which are not feasible to do on sorted macrophages.
• Also, please verify that Pths overexpression is working in the Atossa mutants through qPCR or Western.
As we overexpress Pths just in macrophages in our experiments, qPCR or Western on embryos would not reveal increased levels and sorting macrophages isolated from embryos for Westerns is extremely time consuming and expensive. We therefore imaged fixed atos BGP mutant embryos expressing the HA-tagged Porthos in macrophages and stained them with an anti-HA antibody. As seen in the confocal images the HA-tagged Porthos (shown in green) is found in the nucleus (blue) of macrophages labeled with a cytoplasmic marker in red (Fig. EV4I).
• As an alternative approach, can you rescue the ribosomal assembly defect in Atossa mutants to determine if this is responsible for downstream mitochondrial phenotypes? One could activate mTORC1 (pharmacological or genetic) to rescue mitochondrial defects present in Atossa mutants since mTORC1 controls ribosome assembly, mitochondrial gene translation, and PGC-1a activity. Or perhaps overexpress a ribosomal assembly factor that promotes 40S assembly.
We thank the reviewer for this wonderful suggestion. We examined if we could restore the deficiency of macrophage tissue invasion in atossa-KD or porthos-KD embryos by activating the dTORC1 signaling pathway in macrophages, expressing different RNAis against TORC1 inhibitory components, including Nrpl2, Iml1, and TSC1. We knocked down these different TORC1 suppressors in (Fig 5E,G) atos-KD or (Fig 5F,H) pths-KD macrophages, and found that they could largely restore their germband invasion. This data supports the conclusion that Porthos acts to enhance ribosomal assembly to enable macrophage germband invasion.
This data is discussed in lines 322-331 in the manuscript and shown in Fig 5E-H. • Can you express a mutant of Pths in Pths KD cells that fails to localize to the nucleus to assess whether its function in nucleolar ribosomal assembly is important for downstream phenotypes?
We made a mutant form of Porthos lacking the Nuclear Localization Signal (NLS). We observed that Porthos nlsis mainly in the cytoplasm of S2R+ cells (Appendix Fig S1). However, when we overexpressed the FLAG::HA-tagged Pths nlsin macrophages in atos PBG mutant embryos, while it showed some effect, it was unable to rescue macrophage germband invasion (Appendix Fig S1). Therefore, we conclude that Porthos nuclear localization plays a critical role to help Atossa to facilitate macrophage invasion.
This data is described in lines 313-321 in the manuscript and shown in Appendix Fig S1. • Fig 6. b,c -For graphical representation of OCR in bar graph form, I would subtract nonmitochondrial respiration as a baseline correction.
We have changed the figure accordingly.
On that note, it is odd that your non-mitochondrial respiration is so different between genotypes. Perhaps this indicates a seeding/measurement issue since the cells are semiadherent.
We always seeded the same number of cells. We did not observe changes in the measurements over the time of the experiment making us think it is not likely to be an issue of differential adherence to the probe between the two genotypes. One of Porthos' targets is Lysl oxidase (Appendix Fig S2B), which utilizes oxygen; this could perhaps be an explanation. See lines 417-8.

• Perform either SDS or BN-PAGE analyses for Atossa and Porthos LOFs to confirm nuclear-encoded translation defects.
Unlike in mammalian systems, there are few available antibodies against Drosophila proteins. Thus we were unable to obtain antibodies corresponding to any of the direct targets we identified in the RNAseq. To get around this we utilized commercially available antibodies that had been validated for Drosophila (Teixeira et al 2015 PMID 25915123) against subunits of the OxPhos complexes whose protein levels had been shown to depend on the protein levels of our direct targets. Lower levels of the mammalian ortholog of the predicted complex I assembly factor we identified as a target in the RNAseq lead to reduced levels of other complex I proteins, including MT-ND1 (see Formosa et al., 2015 Fig 2C,D). Similarly, in humans the absence of subunit g of complex V, one of our targets, has been shown to lead to lower protein levels of multiple other subunits including ATP synt-β (see He et al., 2018 Fig 5A). In Western blots we found 73% and 31% lower levels of these CI and CV proteins respectively in pths-KD S2R+ cells compared to the control (CI MT-ND1, p<0.0001; CV ATP synt-β, p=0.03) (shown in Fig 6G-H). To test for a possible general deficiency in protein translation we examined the non-target proteins profilin and β tubulin and found no significant change in levels ( Fig 6I) (profilin, p=0.26; β tubulin, p=0.55). In sum, our results argue that Pths does not affect protein translation generally, but is required for the enhanced levels of a subset of proteins, many of which are involved in mitochondrial and metabolic function.
The above description has been added to the results in lines 364-382 and as new Figure panels as mentioned above.
• Please confirm efficacy of complex III and V knockdowns by qPCR or Western.
We have validated the RNAi-downregulation efficacy of complex III and V by performing qPCR on the respective RNAi-expressing embryos (Fig. EV5E) (lines 437-8).
• Is there any mRNA-mRNA or protein-protein correlation between Atossa and PGC-1a? Is there any overlap between Atossa and PGC1-a targets? This could strengthen arguments that these pathways can act in concert.   Thank you for submitting your revised manuscript (EMBOJ-2021-109049R) to The EMBO Journal. Your amended study was sent back to the three referees for re-evaluation, and we have received comments from two of them, which I enclose below. Please note that we have editorially assessed your response to the concerns pointed to earlier by referee #2 and found these to be satisfactorily responded to. As you will see, the other referees stated that the issues raised have been adequately addressed and they are broadly now in favour of publication, pending a minor revision.
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Thank you for giving us the chance to consider your manuscript for The EMBO Journal. I look forward to your final revision. >> Compile supplemental figures and legends as one 'Appendix' file with a ToC on its first page, save as PDF, rename figures to "Appendix Figure S1" etc.; Add three Suppl. Tables from the other doc, rename to "Appendix Table S1" etc. .
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Further information is available in our Guide For Authors: https://www.embopress.org/page/journal/14602075/authorguide We realize that it is difficult to revise to a specific deadline. In the interest of protecting the conceptual advance provided by the work, we recommend a revision within 3 months (2nd May 2022). Please discuss the revision progress ahead of this time with the editor if you require more time to complete the revisions. Use the link below to submit your revision: The authors have addressed the previous reviewers comments. One key point is that they don't think that the mRNA targets have TISU elements, but do note that they have shorter 5' UTRs (which is a hallmark of tisu mRNAs). They don't see changes in mtor signaling as assessed by p-4ebp1 (although they don't see a phospho ladder as one might expect), but mtor hyperactivity does increase the invasion phenotype. The authors should comment on this butI don't think this should affect their publication success, but does make me wonder how correct they are on these points We thank the reviewer for their critical examination of the manuscript and for their approval of its publication. While TISU element-containing mRNAs have very short 5'UTRs, not all mRNAs with shorter 5'UTRs have TISU elements. TISU element sequences have been found to be enriched in mRNAs with 5' UTRs shorter than 46 bases with a median length of 12 bp (https://doi.org/10.1371/journal.pone.0003094). While the 5'UTRs of the mRNAs whose polysome presence is dependent on Porthos are shorter than that of the average mRNA found in S2 cells, they are not predominantly that short (the median for Porthos targets is 88.5 bp, for all mRNAs 159 bp). We have now added a small table to Figure 5D to highlight this and added this to the Figure legend (line 2237-41).
"The corresponding table shows median/mean 5'UTR lengths in base pairs (bp) for all mRNAs expressed in S2 cells (non-targets) and for the subset whose TE is enhanced by Porthos (Porthos targets)." A phospho ladder for 4EBP1 is not generally observed in Westerns from Drosophila extracts (eg Fig 2A- . Based on known mTOR pathway functions we interpret its hyperactivity as generally increasing ribosomal protein and rRNA levels as well as rRNA processing. This could drive more ribosomal assembly despite the reduced levels of the Porthos helicase whose orthologs aid rRNA processing. The absence of the change in 4EBP1 phosphorylation and the much more specific effect on translation argue that Atossa does not act upstream of mTOR to stimulate its activity. To clarify our description of our interpretation we have now changed the discussion (blue text) to read (lines 615-26): "Enhancing mitochondrial energy production by raising ribosome levels and thereby increasing the translational efficiency of already existing mRNAs is a complementary mechanism to those previously identified. In response to nutrient availability, mTORC1 stimulates all cap-dependent translation and activates the transcription of ribosomal RNAs and proteins and the processing of rRNAs; this ultimately leads to higher levels of many proteins enabling growth, including those required for mitochondrial function (Borregaard and Herlin, 1982). We find that Atossa also affects ribosomal assembly and the atos mutant can be rescued by TOR pathway activation. However the unchanged p-4EBP1 levels and the specific translational effect in the mutants argue that Atossa does not regulate TORC1 activity and that the observed rescue is due to higher general ribosome production. CLUH forms RNA granules, directly binding to, stabilizing and enhancing the translation of mRNAs encoding mitochondrial proteins involved in metabolism, while inhibiting translation of those involved in mitochondrial transcription and translation Thus, I am pleased to inform you that your manuscript has been accepted for publication in the EMBO Journal.
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Is there an estimate of variation within each group of data? For analysis of Drosophila embryos, we generally utilized at least an n of 15, which in our experience lets us detect robust phenotypes. For live imaging we utlized at least n=3. There were no `'animal`' studies.
We only excluded samples that were the wrong stage in our analysis of timed embryo collections, or entire experiments if the antibody staining didn't work even in the control.
Our analysis was conducted on Drosophila embryos collected during a certain time period from mothers of one genotype. There was no subjective bias utilized in which mothers and fathers to point into a cage. We just took all the adults from a certain genotype.

Manuscript Number: EMBOJ-2021-109049
Yes We always tested for a normal distribution before analysis using Prism. This is described in the methods section on statistics.
Yes, we always calculated Standard Deviation and then Standard Error. This is described in the Figure legends and in Dataset EV1 No animal study was conducted in this paper. If what we did above is considered randomization, then we conducted randomization.
We always converted the names of all the images nto a code before we examined them, so that they could be analyzed blind. This is described in the methods section on statistics.
We did blinding.

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